Engagement of the T cell receptor (TCR) initiates a complex signaling cascade that culminates in activation of several key transcription factors, including NF-?B. NF-?B induces expression of a wide array of genes required for T cell proliferation and effector differentiation. We have recently identified the POLKADOTS signalosome, and we have demonstrated that this complex cytoplasmic structure plays an essential role in the TCR-to-NF-?B cascade. The purpose of our proposed studies is to elucidate the molecular mechanisms by which the POLKADOTS signalosome precisely controls activation of NF-?B. This work will be accomplished via collaboration between the groups of Brian Schaefer, who identified the POLKADOTS signalosome, and Wolfgang Losert, who has developed algorithms for quantitative analysis of patterns of protein distribution. By combining the expertise of our respective groups, we expect this work to lead to new hypotheses and conclusions regarding how changes in protein distribution are mechanistically connected to the signaling function of the POLKADOTS signalosome. We will achieve these goals through three Aims: The goal of Aim 1 is to quantitatively define discrete functional domains within POLKADOTS signalosome, to better define how the POLKADOTS signalosome simultaneously controls both Bcl10 signal transmission to NF-?B and proteolysis of Bcl10, limiting NF-?B activation. Our studies in Aim 2 will analyze the contribution of intact, dynamic microtubules to the transport and aggregation of POLKADOTS signalosomes, to determine whether this aggregation process is a mechanism of kinetic regulation of TCR signaling to NF-?B. In Aim 3, we will determine how actin cytoskeletal dynamics regulate selective autophagy of Bcl10, revealing mechanisms controlling the selective proteolysis of Bcl10, and, more generally, mechanisms regulating p62-dependent selective autophagy. Together, we expect these data to yield new mechanistic insights regarding how signalosomes simultaneously coordinate diverse signal regulatory activities. We furthermore predict the innovative analytical methods developed in the course of this research could be readily translated to other signaling systems. Our work will thus suggest new avenues for exploring molecular mechanisms of signal transduction in a wide array of biological pathways important to human health.